Abstract Endogenous rhythmic behaviors are evolutionarily conserved and essential for life. Both in mammalian and invertebrate models, there are known requirements for glial cells, in addition to neuronal circuits, in the regulation of circadian behavior and sleep (reviewed in [1;2]). Using the Drosophila model, our lab was the first to demonstrate that astrocytes were required, in vivo, for normal rhythmic behavior [3;4], and recent studies from other labs demonstrate a conserved role for astrocyte-neuron communication in the circadian systems of mammals [5;6]. Using Translating Ribosome Affinity Purification (TRAP) methods, combined with RNA-seq, we delineated the genome-wide expression patterns of Drosophila astrocytes. Based on this knowledge, we performed genetic screens to identify glial factors that regulate rhythmic behaviors. Those screens identified glial vesicle release components, an SLC-type transporter and several small secreted proteins that are required for normal circadian activity rhythms or sleep [7]. One glial gene encodes an Ig-domain protein with a high-quality signal sequence, indicative of secretion; it is required in astrocytes for the regulation of night sleep. Although astrocytes are often described as a single class of cells, accumulating evidence indicates that fly and mammalian astrocytes as well as other classes of glia are heterogeneous with many different morphological and functional subtypes [8-10]. This application proposes molecular genetic studies to identify glial subpopulations that mediate regulation of circadian behavior or sleep. Aim 1 will employ two alternative genetic methods to define glial cell subpopulations that contribute to rhythmic behavior. Aim 2 will use TRAP-seq techniques to derive the genome-wide expression pattern of glial subpopulations that are relevant for behavior with the goal of defining molecular markers of functional glial cell heterogeneity. Preliminary results documenting feasibility for both aims are included in the proposal. Together, the two aims will spatially localize glial cell subpopulations that regulate rhythmic behaviors and provide molecular markers for the further analysis of these cell populations.